Neuroectodermal tumors are highly malignant, and include neuroblastomas, small cell cancinoma of the lung, gliomas, neuroblastomas and melanomas. Of the neuroectodermal tumors, neuroblastomas occur during infancy and early childhood. Except for Wilms' tumor, they are the most common retroperitoneal tumors in children. Neuroblastomas arise most commonly in the adrenal medulla, but they may also develop in other sympathetic ganglia within the thorax or abdomen. These tumors metastasize early with widespread involvement of lymph nodes, liver, bone, lung and marrow. The prognosis is often good when the tumor is diagnosed prior to obvious metastasis, but with metastasis, prognosis is poor despite the extensive use of radical surgery, deep X-ray therapy, and chemotherapeutic agents.
Several antigenic determinants have recently been detected on neuroblastoma cells with monoclonal antibodies (Mabs). See Seeger, Ann. Intern. Med., 97, 873 (1982); Wikstrand et al., Cancer Res., 42, 267 (1982); Wikstrand et al., J. Neuroimmunlogy, 3, 43 (1982); Eisenbarth et al., Proc. Natl. Acad. Sci. USA, 76, 4913 (1979); Liao et al., Eur. J. Immunol., 11, 450 (1981); Seeger et al., Cancer Res., 4, 2714 (1981); Kennett et al., Advances in Neuroblastoma Research, p. 209, Raven Press, N.Y. (Evans ed.) (1980); Seeger et al., J. Immunol., 128, 983 (1982); Kemshead et al., Pediatr. Res., 15, 1282 (1981). Gangliosides GD3 and GD2 are among the antigenic determinants detected on neuroblastoma cells.
Gangliosides (sialic acid-bearing glycolipids) are thus rapidly being characterized as tumor markers that are relevant target antigens for monoclonal antibody (Mab)-mediated immunotherapy [Dippold et al. (1983) Cancer Res. 44:806-810; Houghten et al. (1985) Proc. Natl. Acad. Sci. USA 82:1242-1246; Hellstrom et al. (1985) Proc. Natl. Acad. Sci., USA 82:1499-1502; Cheresh et al. (1985) Proc. Natl. Acad. Sci. USA 82:5155-5159; Honsik et al. (1985) Natural Immunity and Biological Response 4:253; and Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653-8657].
The disialoganglioside GD3, is expressed preferentially on human melanoma cells [Dippold et al. (1980) Proc. Natl. Acad. Sci., USA 77:6114-6118; Cheresh et al. (1984) Proc. Natl. Acad. Sci., USA 81:5767-5771], and is an effective target in vitro for both complement-mediated tumor cytolysis and antibody-dependent cellular cytoxicity (ADCC) by Mabs of the IgG3 subclass [Hellstrom et al. (1985) Proc. Natl. Acad. Sci., USA 82:1499-1502; Cheresh et al. (1985) Proc. Natl. Acad. Sci., USA 82:5155-5159; and Honsik et al. (1985) Natural Immunity and Biological Response 4:253]. Monoclonal antibodies of the IgG3 class directed against GD3 have been reported to effectively suppress the establishment of human melanoma tumors in the xenotransplant nude mouse mode [Hellstrom et al. (1985) Proc. Natl. Acad. Sci., USA 82:1499-1502; and Cheresh et al. (1985) Proc. Natl. Acad. Sci., USA 82:5155-5159].
Murine mononuclear splenocytes "armed" with anti-GD3 Mabs were recently reported to eradicate well established and progressively growing human melanoma tumors in nude mice [Honsik et al. (1985) Natural Immunity and Biological Response 4:253]. Additionally, Houghten et al. (1985) Proc. Natl. Acad. Sci., USA 82:1242-1246, using Mab R24 (IgG3) directed to GD3 (discussed in U.S. Pat. No. 4,507,391), observed major tumor regressions in 3 of 11 melanoma patients treated with that antibody in a Phase I clinical trial. It was also reported in Dippold et al., Cancer Res., 44, 806 (1984), that Mab R24 could kill GD3-containing human melanoma cells in vitro after prolonged exposure (greater than 24 hours) to the antibody suggesting an additional, as yet undefined, mechanism of tumor cell killing. Taken together, these findings demonstrate that the potential therapeutic efficacy of anti-GD3 Mabs warrants further study of ganglioside as immunotherapeutic targets.
The fact that the GD2 antigen was shown to be heavily expressed on most excised melanoma and SCCL tumors, as well as on numerous tumor cell lines, Pukel et al., J. Exp. Med., 155, 1133 (1982) and yet is virtually absent from most normal tissues, suggests that it might be a good target antigen for in vivo specific immunotherapy, and tumor imaging.
In a recent report by Kipps et al., J. Exp. Med., 161, 1 (1985), using isotype switch variants of a Mab directed to an epitope on Class I human histocompatibility antigens, an IgG2a isotype variant was shown to be more effective in directing ADCC than the corresponding IgG1 or an IgG2b variant. Recent work from our own laboratory, Schulz et al. (1985) J. Exp. Med. 161:1315-1325, also showed specific cytolysis. That work, using Mab 9.2.27, an IgG2a monoclonal antibody that immunoreacts with a chondritin sulfate proteoglycan that is preferentially expressed on human melanoma cells, illustrated that human melanoma tumors, established and progressively growing in nude mice, could be eradicated by simultaneous injection of that Mab along with a relatively large dose of mononuclear splenocytes. Neither the splenocytes nor the antibodies alone achieved significant tumor regression.
Park et al., Cellular Immunol., 84, 94 (1984), reported that a monoclonal antibody of IgG2b isotype could sensitize K562 human erythroleukemia cells to ADCC-mediated lysis. In that case, it was reported that the Mab accelerated killing of the target cells by large granular lymphocytes known to be enriched in natural killer (NK) cells.
Taken together, the results of these studies indicate that monoclonal antibodies may not only be useful reagents for the immunotherapy of cancer, but also that different Mabs can induce tumor killing by several different or even a combination of effector mechanisms.
Some additional recent reports suggest that mouse monoclonal antibodies are relatively well tolerated in humans and pose minimal risks and few, if any, side effects, Oldham et al., J. Clin. Oncol., 2, 1235 (1984). In using murine Mab 9.2.27, discussed before, to treat melanoma patients, that antibody was shown by the above workers to localize specifically to the tumor site with little if any adverse side effects, but provided no apparent clinical improvement of the disease over the period of time of their study in stage four patients with large tumor burdens.
Another form of anti-tumor immunotherapy involves the exposure of mononuclear lymphocytes to the lymphokine interleukin-2 (IL-2) to generate lymphokine activated killer (LAK) cells [Yron et al. (1980) J. Immunol. 125:238-245; Lotze et al. (1981) Cancer Res. 41:4420-4425; Grimm et al. (1982) J. Exp. Med. 155:1823-1841; Grimm et al. (1983) J. Exp. Med. 158:1356-1361].
For example, Rosenberg et al. reported that the use of recombinant IL-2 (rIL-2)-stimulated human LAK cells in phase I clinical trials resulted in marked tumor regression in several patients with progressively growing neoplasms [Rosenberg et al. (1985) New Engl. J. Med. 313:1485-1492]. In those clinical trials, large numbers (10.sup.10 -10.sup.11) of human peripheral blood leukocytes were subjected to doses of rIL-2 (1000 U/1.5.times.10.sup.6 cells/ml) ex vivo for up to 96 hours before being injected intravenously (i.v.) back into the patients. These individuals then received additional i.v. injections of rIL-2 (up to 100,000 U/kg body weight) during the course of their treatment.
Although some dramatic tumor regressions were observed in several patients with extensive neoplasms, the treatment also produced a number of relatively severe clinical problems. Among those clinical problems were fluid retention, pulmonary edema and occasional respiratory distress requiring intubation. Those problems were a result of the direct toxicity effects of rIL-2 infusions.
In a still more recent report, Rosenberg et al. (1986) Science 233:1318-1321, reported use of a three-part modality consisting of (a) an immunosuppressing drug, cyclophosphamide, along with (b) rIL-2-expanded tumor-infiltrating lymphocytes (TIL) obtained from resected tumors and (c) a relatively low dose of rIL-2 to successfully cure twelve mice with MC-38 colon adenocarcinoma of advanced hepatic metastases, and to cure up to 50 percent of mice with advanced pulmonary metastases. The rIL-2 was reportedly administered systemically at 25,000 units three times per day for four days. Those tumors reportedly did not respond to LAK therapy.
Several groups have reported the use of bispecific heteroconjugate (hybrid) antibodies for mediating target cell lysis. For example, Jung et al. (1986) Proc. Natl. Acad. Sci. USA 83:4479-4483 reported effective killing of human melanoma target cells that were coated with a bispecific hybrid antibody composed of one paratopic molecule that immunoreacted with an antigen on the target cell and a second paratopic molecule (OKT3, ATCC CRL 8001) that immunoreacted with human T cells. Human T cells previously stimulated by contact with OKT3 were used as the effector cells.
Bevan, Staerz and co-workers have published several papers dealing with the use of bispecific hybrid antibodies to kill target cells in murine systems. For example, Staerz et al. (1985) Nature 314:628-631 reported use of a hybrid antibody one of whose paratopic portions immunoreacted with an allotypic epitope on the T cell receptor of about 25 percent of peripheral T lymphocytes and the other of whose paratopic portions immunoreacted with the Thy-1.1 alloantigen. That report showed that target S. AKR lymphoma cells that expressed the Thy-1.1 alloantigen were killed by coating the target cells with the bispecific hybrid antibody and subsequent admixture of the coated target cells with various dilutions of cytotoxic T lymphocytes bearing the antigen recognized by the other paratopic portion of the hybrid. Potential target cells that did not bear the Thy-1.1 antigen were not killed. Related studies by that group can be found in Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453-1457; Staerz and Bevan (1986) Eur. J. Immunol., 16:263-270; and Staerz and Bevan (1985) Eur. J. Immunol., 15:1172-1177.
Still further, Perez et al. (1986) J. Exp. Med. 163:166-178 described a bispecific heteroconjugate antibody that reportedly targets rIL-2-activated peripheral blood mononuclear cells (PBMCs) to tissue. Thus, one portion of that heteroconjugate binds through its paratope [Fab fragment of Mab OKT3 (ATCC CRL 8001)] to the activated PBMCs, while the other paratope of the heteroconjugate (Fab fragment of a target-binding Mab) binds to the target tissue.
In their study, Perez et al. depleted the lymphocyte effector cell population of monocytes as well as Leu-11-positive (Leu 11.sup.+) cells. They reported that their IL-2-activated effectors exhibited the T8 antigen (were T8.sup.+) and that elimination of T8.sup.+ cells using Mab OKT8 (ATCC CRL 8014) and complement eliminated cytoxicity. Removal of T4.sup.+ cells by a similar strategy [Mab OKT4 (ATCC CRL 8002) plus complement] increased lysis in the presence and absence of IL-2 activation. Those authors also reported the loss of about one-half of the lytic activity when activated, hybrid antibody-coated effectors were maintained free of IL-2 between 8 and 24 hours.
Bispecific heterobifunctional monoclonal paratopic molecules and methods of their preparation are also disclosed in U.S. Pat. No. 4,444,878 and PCT/US82/01766 (WO 83/02285), whose disclosures are incorporated herein by reference. The techniques described in U.S. Pat. No. 4,350,626 for linking Fab portions of anti-tumor antibodies to the ricin A subunit can also be utilized for linking Fab portions of desired antibodies in the preparation of bispecific hybrids.